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WO2022103126A1 - Vaccin à coronavirus utilisant un adénovirus inapte à la réplication qui exprime simultanément une protéine spike de coronavirus et une protéine de nucléocapside - Google Patents

Vaccin à coronavirus utilisant un adénovirus inapte à la réplication qui exprime simultanément une protéine spike de coronavirus et une protéine de nucléocapside Download PDF

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WO2022103126A1
WO2022103126A1 PCT/KR2021/016258 KR2021016258W WO2022103126A1 WO 2022103126 A1 WO2022103126 A1 WO 2022103126A1 KR 2021016258 W KR2021016258 W KR 2021016258W WO 2022103126 A1 WO2022103126 A1 WO 2022103126A1
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sars
protein
coronavirus
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최정우
정광일
최강석
손승은
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BioLeaders Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a recombinant adenovirus in which the spike protein and the nucleocapsid protein of the coronavirus are inserted into the E1 region and the E3 region of the replication-deficient adenovirus, and a coronavirus vaccine using the same. .
  • Coronavirus disease 2019 (hereinafter referred to as “COVID-19”) is a new type of coronavirus (hereinafter referred to as “SARS-CoV- 2”)), which is transmitted when droplets (saliva) of an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. Respiratory symptoms such as cough or shortness of breath and pneumonia are the main symptoms, but the frequency of asymptomatic infections is also high.
  • SARS-CoV-2 has an envelope and has a single-stranded RNA of about 30 kb as a genome.
  • the gene encodes structural proteins such as a nucleocapsid (N) protein, a membrane (M) protein, an envelope (E) protein, and a spike (S) glycoprotein ( FIG. 1 ).
  • N nucleocapsid
  • M membrane
  • E envelope
  • S spike glycoprotein
  • the spike protein is known to play an important role in the initiation of infection in relation to the attachment to the surface of the host cell.
  • the S1 domain of the spike protein is a receptor binding domain (RBD) that mediates specific binding to cell receptors.
  • RBD receptor binding domain
  • a nucleocaptid protein is a protein involved in the packaging and replication process of the viral genome, and consists of two viral RNA binding sites linked by a linkage region, and the linkage region is serine/ Arginine is abundant and known to be a conserved fraction (Sebastano et al., 2020).
  • SARS-CoV-2 vaccine is being developed in a primate model based on SARS-CoV-2 research, and the SARS-CoV-2 vaccine under development has been shown to increase humoral and cellular immune responses through inoculation ( 2).
  • some pharmaceutical companies are currently pursuing the development of a SARS-CoV-2 vaccine, but as it is still in the development stage, it is necessary to verify the effectiveness through preclinical and clinical trials.
  • SARS-CoV-2 vaccine currently used is made based on information on the Wuhan-hu virus strain, and is not specialized to protect against SARS-CoV-2 mutant virus, SARS-CoV- 2
  • SARS-CoV- 2 There is an urgent need to develop a next-generation vaccine that can effectively prevent the spread of mutated viruses and can be used universally.
  • the present invention provides that a spike protein of a coronavirus (SARS-CoV-2) is inserted into the E1 region of a replication-deficient adenovirus, PgsA and a nucleo of a coronavirus (SARS-CoV-2)
  • a spike protein of a coronavirus SARS-CoV-2
  • PgsA a replication-deficient adenovirus
  • SARS-CoV-2 a nucleo of a coronavirus
  • the present invention also provides a recombinant adenovirus comprising the amino acid sequence of SEQ ID NO: 1 in the E1 region and the amino acid sequence of SEQ ID NO: 2 in the E3 region.
  • the present invention also provides a coronavirus (SARS-CoV-2) vaccine produced by the recombinant adenovirus.
  • SARS-CoV-2 coronavirus
  • the present invention also provides a coronavirus vaccine characterized in that it acts on a SARS-CoV-2 mutant virus.
  • the present invention also provides a coronavirus vaccine characterized in that it acts on the D614G mutant virus.
  • the present invention also provides a coronavirus vaccine, characterized in that it further comprises an adjuvant.
  • the vaccine composition according to the present invention can induce an immune response against SARS-CoV-2 by inducing a humoral immune response and a cell-mediated response.
  • 1 shows the SARS-CoV-2 structure and a gene encoding the same.
  • Figure 2 shows the humoral and cellular immune responses through vaccination.
  • S protein SARS-CoV-2 spike protein
  • PgsA SARS-CoV-2 nucleocapsid protein
  • N protein SARS-CoV-2 nucleocapsid protein
  • FIG. 4 shows the expression of spike protein and nucleocapsid protein in cells infected with a recombinant adenovirus according to the present invention.
  • FIG. 5 shows the expression levels of CD80, CD86 and MHCII by the adenovirus recombined according to the present invention.
  • FIG. 6 is a graph showing the antibody titer to the spike antigen of the vaccine according to the present invention.
  • FIG. 7 is a graph showing the evaluation of stability of vaccine immunogenicity formation according to the present invention.
  • 9 is a graph relating to the memory capacity of cytotoxic T cells to the spike antigen.
  • 10 is a graph relating to the memory capacity of helper T cells for the spike antigen.
  • 11 is a graph showing that a cell-specific immune response was induced by the spike antigen.
  • FIG. 12 is a graph showing the antibody titer to the nucleocapsid antigen of the vaccine according to the present invention.
  • 13 is a graph showing the memory capacity of cytotoxic T cells to the nucleocapsid antigen.
  • FIG. 14 is a graph relating to the memory capacity of helper T cells for the nucleocapsid antigen.
  • 15 is a graph showing that a cell-specific immune response was induced by a nucleocapsid antigen.
  • 16 and 17 confirm the antigen-antibody reaction that occurs between the antigen expressed in the cells infected with the recombinant adenovirus according to the present invention and the antibody of a cured patient.
  • 18 is a graph showing the humoral immunity of the vaccine according to the present invention to SARS-CoV-2 D614G mutant virus.
  • the spike protein of the coronavirus is inserted into the E1 region of the replication-deficient adenovirus, PgsA and the coronavirus (SARS-CoV-2) It provides a recombinant adenovirus in which the nucleocapsid protein is expressed in a form inserted into the E3 region of the replication-deficient adenovirus.
  • the spike protein consists of a membrane-distal S1 subunit and a membrane-proximal S2 subunit and exists as a homotrimer in the viral envelope, where the S1 subunit recognizes receptors through its receptor binding domain and the S2 subunit is required for viral entry.
  • This spike protein which is responsible for membrane fusion, essentially acts upon binding to a host cell receptor, and is known as a factor determining the range of a host to infect.
  • Nucleocapsid is composed of nucleocapsid protein bound to viral RNA. Nucleocapsid protein binds to viral RNA genome and plays an important role in stabilizing and packaging the genome during viral particle assembly, envelope formation, and RNA synthesis. , Since the nucleocapsid protein has a low mutation rate, when the nucleocapsid protein is used as an antigen, it can effectively act on SARS-CoV-2 mutant virus.
  • the present invention provides a recombinant adenovirus comprising the E1 region comprising the amino acid sequence of SEQ ID NO: 1 and the E3 region comprising the amino acid sequence of SEQ ID NO: 2.
  • SARS-CoV-2 vaccine prepared by recombinant adenovirus.
  • the protein may be a polypeptide having the amino acid sequence of SEQ ID NOs: 1 and 2.
  • the protein has 80 to 99%, 85 to 99%, preferably 90 to 99% sequence homology therewith, not only of the polypeptide identical to SEQ ID NOs: 1 and 2, but also of a polypeptide in which amino acids are substituted by conservative substitution. It may include all polypeptides having
  • the present invention provides a coronavirus vaccine characterized in that it acts on a SARS-CoV-2 mutant virus, wherein the SARS-CoV-2 mutant virus may be a D614G mutant virus.
  • the term “vaccine composition” refers to a composition containing at least one or more immunologically active ingredients that induce an immunological response in an animal.
  • the term “antigen” refers to a protein expressed by the virus as a component capable of inducing an immune response among the components of the virus.
  • antibody used in the present invention is a component produced by antigen stimulation in the immune system, and is a protein that specifically binds to a specific antigen and circulates in the lymph and blood and causes an antigen-antibody reaction.
  • Antigen-antibody reaction has high specificity for each antigen, and when antibodies are made by B cells of lymphocytes, antibodies produced by specific antigens do not react with other antigens in principle. This high specificity is used for tests such as immunity, allergy, and determination of the type and type of various diseases and infections.
  • the vaccine composition may be in any form known in the art, for example, in the form of solutions and injections, or in solid form suitable for suspension, but is not limited thereto. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or may be prepared in the form of an aerosol or spray. They may also be incorporated into transdermal patches. In the case of a solution or injection, if necessary, it may contain propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis.
  • the vaccine composition of the present invention may include a pharmaceutically acceptable carrier or diluent.
  • Suitable carriers for vaccines are known to those skilled in the art and may include, but are not limited to, proteins, sugars, and the like.
  • Such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous carriers may be propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • the vaccine composition may further include an adjuvant (adjuvant, immune-enhancing agent, immune-enhancing agent).
  • adjuvant refers to a compound or mixture that enhances the immune response and/or promotes the rate of absorption after inoculation, and may include any absorption-promoting agent.
  • Acceptable adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, plurone polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyholimpet he It may include, but is not limited to, mocyanine, dinitrophenol, and the like.
  • the vaccine composition of the present invention may be administered via any one administration route selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal, preferably administered by injection. Do.
  • SARS-CoV-2 infection disease is a disease caused by infection with SARS-CoV-2 virus, which may cause sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia, diarrhea, etc. not limited
  • the vaccine composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount sufficient to exhibit a vaccine effect, and not to cause side effects or serious or excessive immune response, and the level of effective dose is determined by the disorder to be treated. , the severity of the disorder, the activity of the particular compound, the route of administration, the rate of removal of the protein, the duration of treatment, the drug used in combination with or concurrently with the protein, the age, weight, sex, diet, general health condition of the subject, and known in the medical arts. It may depend on a variety of factors, including factors.
  • the SARS-CoV-2 spike ( spike, S) protein-coding gene is inserted, and PgsA and SARS-CoV-2 nucleocapsid (N) proteins, which are proteins with membrane anchoring ability, are inserted in the E3 region of replication-defective adenovirus.
  • PgsA and SARS-CoV-2 nucleocapsid (N) proteins are proteins with membrane anchoring ability, are inserted in the E3 region of replication-defective adenovirus.
  • PgsA serves to enable expression of the nucleocapsid protein in the cell membrane, and the inserted genes are codons optimized for mammalian cell expression, and the protein expressed thereby is It has the ability to inhibit SARS-CoV-2 virus infection by inducing a humoral immune response and a cell-mediated response.
  • candidate groups 1 to 4 (#1, #2, #3, #4) prepared to observe the expression of each protein (antigen) were infected with HEK293 seeded with 5x10 5 cells in 6well. After 48 hours, the expression was observed by western blot using the spike antibody and the PgsA antibody (anti PgsA) (Fig. 4). Among them, the candidate group 3 (#3) with the best expression was used A follow-up experiment was conducted, and the sequence of candidate group 3 is as follows.
  • amino acid sequence of adenovirus E1 into which the gene encoding the SARS-CoV-2 spike protein is inserted is shown in SEQ ID NO: 1 below.
  • amino acid sequence of adenovirus E3 into which genes encoding PgsA and SARS-CoV-2 nucleocapsid proteins are inserted is shown in SEQ ID NO: 2 below.
  • B7 protein is a peripheral membrane protein that appears in activated immune cells. It interacts with T-cell surface proteins, CD28 or CD152, to produce a costimulatory signal that increases or decreases MHC-TCR signaling between immune cells and T cells.
  • B7 proteins There are two main types of B7 proteins, B7-1 (CD80) and B7-2 (CD86), and CD28 and CTLA-4 interact with both CD80 and CD86, respectively.
  • group II major histocompatibility complex II (MHCII) molecules play a role in exposing and expressing antigens to the outside after decomposition into fragments within the cells when foreign antigens are introduced into immune cells.
  • CD86 and MHCII can be evaluation indicators of antigen delivery ability.
  • the antibody titer is the titer of the antibody corresponding to a specific antigen, a unit indicating the amount of antibody, and is expressed as the reciprocal of the maximum dilution concentration at which a reaction occurs when the same amount of antigen is added after diluting a solution containing the antibody.
  • mice using 5 female BALB/c mice per group (Orient, Korea) 6 to 8 weeks old mice, 5x10 8 , on days 0 and 14, 5x10 9 .
  • 5x10 10 VP viral particles
  • SARS-CoV-2 spike protein-specific antibody response was measured through an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the plate was washed with PBST (PBS+0.05% tween20), blocked with 1% BSA (Bovine Serum Albumin, fetal bovine serum albumin) in PBS for 1 hour at room temperature, and then serum was treated with the blocking buffer 1 /800, 1/3200, 1/12800, 1/51200 diluted. After that, 100 ⁇ L of the sample was smeared on each well, incubated for 1 hour at room temperature, washed 4 times with 400 ⁇ L of PBST, diluted with mouse IgG-HRP at a 1:1000 ratio, spread on a plate in 100 ⁇ L, and 1 at room temperature incubated for hours.
  • PBST PBS+0.05% tween20
  • BSA Bovine Serum Albumin, fetal bovine serum albumin
  • mice were immunized with mock and SARS-CoV-2 vaccines via intramuscular inoculation at 1x10 10 VP on days 0 and 14, and after sera was isolated from mice at 2 weeks, 4 weeks, and 6 weeks, enzyme immunoassay (enzyme -linked immunosorbent assay; ELISA) was used to measure the SARS-CoV-2 spike-specific antibody response.
  • enzyme immunoassay enzyme -linked immunosorbent assay
  • a microtiter plate (Nunc, Denmark) was coated with 100 ⁇ L of the spike protein at 100 ng/well in 50 mM sodium bicarbonate buffer (pH 9.6), and incubated at 4°C overnight. The plates were then washed with PBST (PBS+0.05% tween20) and blocked with 1% BSA (fetal bovine serum albumin) in PBS for 1 hour at room temperature. After diluting the serum to 1/800, 1/3200, 1/12800, and 1/51200 with the blocking buffer, 100 ⁇ L of the sample was spread on each well and incubated for 1 hour at room temperature.
  • PBST PBS+0.05% tween20
  • BSA fetal bovine serum albumin
  • the neutralizing antibody means that the antibody binds to the surface structure of the pathogen and inhibits their binding to cells, thereby mediating immunity through so-called neutralization, which blocks infection, suppresses proliferation, and eliminates the pathological effects of toxins. .
  • mice In order to evaluate the neutralizing ability of the SARS-CoV-2 vaccine according to the present invention, five female BALB/c mice per group (Orient, Korea) 6 to 8 weeks old were used. Mice were immunized with mock and SARS-CoV-2 vaccines via intramuscular inoculation with 1x10 10 VP on days 0 and 14, and serum of mice was isolated 3 weeks after final administration. Neutralization capacity was measured using an ELISA kit that detects anti-SARS-CoV-2 neutralizing antibody that inhibits the binding of SARS-CoV-2 viral glycoprotein S1 RBD to cell surface ACE2 receptors.
  • Serum was diluted to 1/2, 1/8, 1/32, 1/128, 1/512, 1/1024, 1/16384, respectively, and reacted with the RBD-HRP conjugate in the kit, and neutralizing antibody against RBD in the serum When present, it forms a bond with RBD-HRP.
  • the microplate is coated with human ACE2 recombinant antigen, and the pre-reacted mixture is loaded onto the plate. In the absence of neutralizing antibody in the mixture loaded on the plate, RBD-HRP binds to ACE2, but in the presence of neutralizing antibody, binding of RBD-hACE2 is inhibited.
  • the HRP substrate solution was added and reacted in the dark for 10 minutes, the reaction stop solution was added, and the absorbance value at 450 nm was measured with a plate reader (multiskansky, manufactured by Thermoscientific) (Fig. 8). ).
  • the inhibition rate was calculated using the negative control solution in the kit, and the average value of the three absorbances was calculated by loading in triple.
  • Inhibition rate (%) 1-((Average absorbance of sample / absorbance of negative control solution) * 100)%
  • the neutralizing antibody titer of the serum corresponding to 50% of the inhibition rate was about 400, confirming the neutralizing ability of the SARS-CoV-2 vaccine according to the present invention.
  • single cells were isolated from the spleen of 6 to 8-week-old female BALB/c mice (Orient, Korea) and then immunized from T cells through flow cytometry. The secretion levels of various cytokines important in inducing a response were measured.
  • mice per group were immunized with mock and SARS-CoV-2 vaccines at 1x10 10 VP on days 0 and 14 through intramuscular inoculation, and 3 weeks later, the spleens of mice were extracted and splenocytes (splenocytes) were used. ), seeded at 1 x 10 6 cells per well in a 24-well plate, and restimulated with 20 ⁇ g/mL of SARS-CoV-2 spikes for 24 hours. After that, a protein transport inhibitor, GolgiPlug (BD Biosciences) was added to accumulate cytokines in the cytoplasm, and cultured for 6 hours.
  • GolgiPlug BD Biosciences
  • the cells were passed through a BD Cytofix/cytoperm kit (BD Biosciences) and stained with anti-CD4, CD8, CD45, IFN-gamma and GranzymeB (BD Biosciences).
  • anti-CD4 and CD45 for CD4 cells co-expressing IFN-gamma and GranzymeB in the gating were measured for cell-related fluorescence levels using a Beckman Coulter naviosflow cytometer.
  • cells co-expressing IFN-gamma and GranzymeB in the cells in the gating were measured for cell-related fluorescence levels using a naviosflow cytometer manufactured by Beckman Coulter (Figs.
  • the IFN-gamma ELISPOT response specific to the spike antigen induced by the SARS-CoV-2 vaccine according to the present invention is shown in FIG. 11, and a specific cellular immune response only by immunization with the SARS-CoV-2 vaccine It can be confirmed that this has been induced. Specifically, five mice per group were immunized with mock and SARS-CoV-2 vaccines at 1x10 10 VP on days 0 and 14 through intramuscular inoculation, and then 3 weeks later, the spleens of mice were harvested to obtain splenocytes.
  • the antibody titer against the nucleocapsid was verified.
  • 5 female BALB/c mice per group Orient, Korea
  • 6 to 8 weeks old mice were immunized with mock and SARS-CoV-2 vaccines by intramuscular inoculation with 1x10 10 VP on days 0 and 14.
  • the mouse serum was isolated and the nucleocapsid-specific antibody response was measured by enzyme-linked immunosorbent assay (ELISA).
  • a microtiter plate (Nunc, Denmark) was coated with 100 ⁇ L of the spike protein at 100 ng/well in 50 mM sodium bicarbonate buffer (pH 9.6), and incubated at 4°C overnight. Plates were washed with PBST (PBS+0.05% tween20), blocked with 1% BSA (fetal bovine serum albumin) in PBS for 1 h at room temperature, and serum 1/800, 1/3200, 1/12800, 1/51200 After dilution with the blocking buffer, 100 ⁇ L of the sample was spread on each well and incubated for 1 hour at room temperature.
  • PBST PBS+0.05% tween20
  • BSA fetal bovine serum albumin
  • single cells were isolated from the spleen of 6 to 8-week-old female BALB/c mice (Orient, Korea), and then T cells through flow cytometry. The secretion levels of various cytokines important in inducing an immune response were measured. Five mice per group were immunized with 1x10 10 VP at 0 and 14 days through intramuscular inoculation with mock and SARS-CoV-2 vaccines. After 3 weeks, the spleens of mice were harvested and splenocytes were isolated.
  • a 24-well plate was seeded at 1 x 10 6 cells per well, and restimulated with 20 ⁇ g/mL of nucleocapsid protein for 24 hours. Then, a protein transport inhibitor, GolgiPlug (BD Biosciences) was added to accumulate cytokines in the cytoplasm, and cultured for 6 hours. After washing with PBS, the cells were passed through a BD Cytofix/cytoperm kit (BD Biosciences) and stained with anti-CD4, CD8, CD45, IFN-gamma, and GranzymeB (BD Biosciences).
  • mice immunized with the SARS-CoV-2 vaccine according to the present invention were significantly increased compared to the control group. This means that when vaccinated mice are infected with SARS-CoV-2 virus, T-cells in the body are activated to secrete IFN-gamma and GranzymeB that can kill foreign factors.
  • the IFN-gamma ELISPOT response specific to the nucleocapsid antigen induced by the SARS-CoV-2 vaccine according to the present invention is shown in FIG. 15, and specific cellularity only by immunization with the SARS-CoV-2 vaccine It can be confirmed that an immune response is induced. Specifically, five mice per group were immunized with mock and SARS-CoV-2 vaccines at 1x10 10 VP on days 0 and 14 through intramuscular inoculation, and then 3 weeks later, the spleens of mice were harvested to obtain splenocytes.
  • FIG. 15 it can be observed that the number of cells (spot number) producing antigen-specific IFN gamma in the spleen of the vaccinated mouse than in the mouse immunized with the mock increases significantly, which is according to the present invention.
  • T-cell is generated by the memory for the vaccine antigen, which can be interpreted as an antigen-specific cellular immune response to the secondary stimulus.
  • mice 6 to 8 week old female BALB/c mice (Orient, Korea) were used. Five mice per group were immunized via intramuscular inoculation with mock and SARS-CoV-2 vaccines at 1x10 10 VP on days 0 and 14, then serum from mice was isolated on day 28, followed by enzyme-linked immunosorbent assay (enzyme-linked immunosorbent). assay; ELISA) to measure the spike D614G-specific antibody response.
  • a microtiter plate (Nunc, Denmark) was coated with 100 ⁇ L of the spike protein at 100 ng/well in 50 mM sodium bicarbonate buffer (pH 9.6), and incubated at 4°C overnight.
  • PBST PBS+0.05% tween20
  • BSA fetal bovine serum albumin
  • the reaction was stopped with 2N sulfuric acid solution, and the absorption capacity at 450 nm was measured using a microplate reader (Multiskansky manufactured by Thermoscientific).
  • the antibody titer was determined with reference to Cutoff value: the mean optical density values at 450nm (OD450)+3 ⁇ standard derivations (SD) from the sera of non-vaccinated animals (Nat Commun. 2020; 11: 4207), and the results were As shown in FIG. 18 , it was confirmed that the vaccine according to the present invention exhibited a stable antibody titer to the Spike D614G mutant virus.

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Abstract

La présente invention concerne : un adénovirus recombinant dans lequel des protéines spike de coronavirus, des protéines de nucléocapside et PgsA sont exprimés sous forme insérée dans la région E1 et la région E3 d'un adénovirus inapte à la réplication ; et un vaccin à coronavirus l'utilisant. Une composition de vaccin selon la présente invention peut induire des réponses immunitaires au SARS-CoV-2 et un virus mutant correspondant par induction d'une réponse immunitaire humorale et d'une réponse à médiation cellulaire.
PCT/KR2021/016258 2020-11-10 2021-11-09 Vaccin à coronavirus utilisant un adénovirus inapte à la réplication qui exprime simultanément une protéine spike de coronavirus et une protéine de nucléocapside Ceased WO2022103126A1 (fr)

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KR10-2021-0152517 2021-11-08
KR1020210152517A KR102399308B1 (ko) 2020-11-10 2021-11-08 코로나바이러스의 스파이크 단백질 및 뉴클레오캡시드 단백질을 동시발현하는 복제불능 아데노바이러스를 이용한 코로나바이러스 백신

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